Inoculation device and method



United States Patent 3,205,151 INOCULATION DEVICE AND METHOD William L. Landau and Ernest R. Burkholder, Chicago, Ill., assignors to Hollister Incorporated, a corporation of Illinois Filed Apr. 17, 1962, Ser. No. 188,166 21 Claims. (Cl. 195-1035) This invention relates to devices useful for inoculating test media with organisms or microorganisms. This invention further relates to the use of such devices.

It is customary in conducting inoculations of test media with organisms or microorganisms to transfer such organisms on a needle, or Wire-like loop or the like to the test media or medium. The common practices result in consumption of valuable working time and also tend to reduce the accuracy of the results. As an example of common practice for identification of a microorganism, a colony (grown on a suitable solid medium) of the organism to be examined is touched with a flame-sterilized and cooled wire needle. The organism adhering to the needle is then emulsified in liquid, from which various tubes containing different media are then inoculated. Another commonly employed method is to inoculate the various media directly from the colony. In the latter procedure, since the wire needle is usually sterilized between each transfer, it is often necessary to use multiple colonies which one assumes to be identical.

It is an object of this invention to provide a new inoculation device which is useful for the eflicient inoculation of a single test material or a plurality of test materials.

Another object of this invention is to provide an inoculation device having a container for a test material or medium and having an inoculation instrument extending through the container and withdrawable from the container so that the inoculum can be carried on the inoculation instrument through the test medium, thereby inoculating during withdrawal of the inoculation instrument through the container.

It is a further object of this invention to provide an inoculation device having a plurality of test medium containers and an elongated inoculation instrument extending through the plurality of containers and withdrawable therefrom for inoculation purposes.

It is still another object of this invention to provide test medium containers which can be connected to each other to form a linear array of such containers through which there can be passed an elongated inoculation instrument for inoculation of test media within the containers.

An additional object is to provide such inoculation devices with covers for the projections of the inoculation instrument therefrom.

Other objects will be apparent from the following descriptions and with reference to the drawings.

In the drawings:

FIGURE 1 is a cross-sectional view of an embodiment of this invention;

FIGURE 2 is an exploded perspective of the embodiment of FIGURE 1; and

FIGURE 3 is a cross-sectional view of another embodiment of the present inoculation device.

With reference to FIGS. 1 and 2 an inoculation device is provided in the form of an elongated tube made up of individual similar molded plastic containers 11 joined in series. Covering the top of tube 10' is a top cap 12. Bottom cap 13 is provided over the bottom of tube 10.

Each of containers 11 has a cylindrical side Wall 14 and a bottom wall 16. A ring projection 17 projecting from each bottom wall 16 is force-fitted within the side wall 14 of another such container 11 from its open top as illustrated in FIG. 1. FIG. 2, an exploded view of the plurality of containers 11, also shows the ring projections 17 which are force-fittable within side walls 14. Thus the bottom of each container 11 is capable of enclosing another such container 11, aligned and forcefitted therebelow. The uppermost container 11a which encloses enclosure 40 is covered by container cap 18 having flanges 17a force-fittable with the open end of the container 11a.

An opening 21 is provided in each bottom wall 16 of each container 11 and an additional opening 22 is provided in container cap 18. The containers 11 define enclosures 40, 41, 42, 43 and 44 containing respectively nutrient media 30, 31, 32, 33 and 34. Inoculation needle 20 is slide-fitted through opening 22 and openings 21 and through the nutrient medium in each enclosure. An inoculating needle 20 has a sample or specimen end 23 and a handle 24 on the opposite end. Caps 12 and 13 enclose the handle and specimen end of needle 20.

The embodiment of FIG. 1 is illustrated as retained within a test tube holder 50 and resting thereon by means of flange 12a around top cap 12.

In FIG. 3 there is illustrated an embodiment having only one container 111 enclosing one enclosure 140 which contains one mass of nutrient material 130'. Needle having bent wire handle 124 and specimen end 123 is capable of being slidably mounted through opening 122 and opening 121 with container cap 118 moved into position to cover enclosure 140 by forcefitting of flange 117 within container 111. It should also be appreciated that the flange 117 may be threadedly received in the container 111. With cap 118' in position on container 111 and needle 120 passing through cap 118, enclosure 140, nutrient material contained within enclosure and bottom 116, caps 112 and 113 are placed over the ends of the assembly to enclose handle 12 i and specimen end 123.

Referring again to FIG. 1, medium 30 is agar gel con taining dextrose; medium 31 is agar gel containing xylose; medium 32 is agar gel containing lactose; medium 33 is agar gel containing lead acetate; and medium 34 is agar gel containing citrate. Each medium also contains a bacteriological indicator such as phenol red which results in a color change when fermentation occurs. Important by-products of fermentation such as gas may also be observed by bubbles trapped in the agar gel.

In assembling the device illustrated in FIG. 1 each container 11 is sterilized and then filled to the desired level with the respective sterilized molten agar medium. After permitting the molten agar to set, the containers 11 are assembled in alignment to form tube 10 and cap 18 is placed over the uppermost container. Needle 20 is sterilized and introduced through opening 22 and through the plurality of openings 21 until it protrudes from the opening 21 of the bottommost container 112. Caps 12 and 13, also sterilized, are then placed over tube 10.

In use with reference to the embodiment of FIG. 1, cap 13 is removed and specimen end 23 of the needle 20 is contacted with the colony desired to be tested; in this example a colony of bacteria is to be tested for the purpose of determining fermentation properties. Cap 13 is then replaced, cap 12 is removed and needle 2i) is withdrawn by pulling handle 24 upward. As the needle passes through each enclosure it inoculates the medium therein with the same organisms, i.e., organisms from the same colony. After complete withdrawal of needle 20 from opening 22, cap 12 is replaced and tube 10 is then placed in a controlled temperature environment, e.-g., by supporting in an upright position by such means as test tube holder 50. After an adequate incubation period, tube is examined for growth evidenced by cloudiness of the medium, and for fermentation by change in color of the acid-base indicator, i.e., for example, from red to yellow indicating fermentation in the nutrient The walls 14 are of sutficiently transparent plastic material or glass so that the results of fermentation can be observed through the Walls. Where the walls are not transparent, e.g., made of opaque plastic, aluminum, stainless steel, etc., the containers may be disassembled and the media examined. Glucose is included in media 34) in the illustrated embodiment in the last section to be inoculated as a precaution to ensure that the bacteria has passed entirely through the tube and was present for inoculation of each medium therebelow; glucose will be fermented and acidified by almost any bacaterium and so its presence in the last stage acts as a safety valve.

Turning now to the embodiment of FIG. 3, medium 130 is placed within enclosure 140 and the device is assembled in the same manner as described with reference to FIG. 1. In use, the bottom cap is removed, the needle inoculated at specimen end 123 and the needle is withdrawn to inoculate medium 130, whereupon caps 112 and 113 may be replaced.

The top caps 12 and 112 and bottom caps 13 and 113 of the embodiments illustrated not only serve to maintain the ends of the needle sterile and insure against contamination of media within the enclosures, but also serve an important function after the needle has been withdrawn to inoculate the media in that replacing the top 'and bottom caps protects against undue exposure of Workers to microorganisms which may have been deposited at the bottom opening 21 of the container lle and opening 22.

Although we have hereinabove, with reference to the figures, described specific embodiments of the present invention, we do not intend to be limited thereto. Other modifications of the present invention, both as a device and in the use of that device, will be apparent to those having ordinary skill in the art.

Although preferably presterilized media are added to presterilized containers and after solidification of the media, the containers are joined in the tube shape and a sterile wire inoculating needle is passed through the entire length, alternatives in assembling the devices described herein will be apparent. For example, the containers may be loosely assembled on the sterile wire inoculating needle such as is illustrated in FIGUREZ and the media added to the containers. After solidification of the media or even before solidification of the media, the containers may then be joined to form the tube configuration.

Although embodiments of our present device have been described which basically provide one or more containers for the test medium, which containers have openings in opposing walls for penetration of the inoculation instrument, any containers having penetrable walls may be used. For example, a puncturable material such as a polyester resin plastic material may be used to form the containers, and a puncturing inoculation instrument, e.g., a needle or knife or blunt stiff wire, may be urged through the walls to create the openings at the time of assembly. Additionally, regardless of the wall material, other inoculation instruments other than sharp and bluntend inoculation wires or needles may be used, e.g., knife, fiber strips such as flexible and rigid plastic strips, etc., preferably elongated.

The test medium may be any microorganism-affecting substance such as an antibiotic, phage or assi-m-ilable carbon or nitrogen containing compound, which affects the growth pattern of at least one micro-organism. The effect obtained within the device may be one or inhibition or enhancement and includes the use of bactericidal, bacteriostati'c, and nutrient substances. Such test media and other test media are well known to the art and are fully intended for u e in accordance herewith. Te t media are 4- often specially adapted, by specific additions, to a group of bacteria or other microorganism which is to be detected, in that substances promoting the growth or the inhibition of this group of microorganisms are contained within the test media. Included within such additives are also substances with an acid or basic reaction, which permit a predetermined pH value to be produced in the nutrient medium, the pH value being of use in the development of certain types of bacteria. Such specially prepared nutrient media are common in the art and used in bacteriological work of all types.

As indicated above, the test medium is intended to include nutrient medium which either prevents the development of certain types of bacteria or which, owing to the metabolic process of the bacteria, undergo chemical changes which can in some Way be observed or measured. The test medium may include dye stuffs which show a clearly detectable change in color due to a variation of the pH value or due to other chemical influence on the dye stuff. It is also possible in accordance herewith to study test organisms, the development of which may be promoted or stopped by certain media; test media for such use containing additives adapted to conducting such studies are well known and conventional.

As more specific examples of such additives which may be included within test media for specific applications, in studying at test organism, various well known chemical bacteriost-atic, or bacteriocidal agents or for promoting growth a variety of basal media with additions of specific amino acids, vitamins or other growth-promoting agents may be used. For example, biotin may be added to stimulate growth. In the detection of typhoid germs, there may be used an aqueous composition containing digested skim milk in a major amount containing smaller amounts of dextrose, dry sodium phosphate, bismuth sulfite, ammonium citrate, sodium sulfite, ferroammonium sulfate, and a color indicator and inhibitor such as a Very small amount of brilliant green. In the detection of diphtheria bacteria, one may use meat extract, common salt, peptone, and a major amount of beef serum, with small amounts of potassium tellurite. Other specific additives and media will be apparent to those in the art.

Although the examples with reference to the drawing used agar as a base for the nutrient material, it is to be understood that other base materials may also be used, including absorbent materials which may be formed of heavy blotting paper, e.g., for use with aqueous solution. Other usable gelling substance, in addition to agar, are gelatinous silica and gelatine. Microorganisms which can be tested using the present device are well known in the art. Microorganisms include bacteria and other viable microorganisms e.g., viruses, fungi, etc.

It is apparent from the foregoing that in the present device only small amounts of the nutrient media need be employed. The small amounts of test medium, in addition to resulting in economy of test medium, enable quicker identification of organisms. A further advantage results from the increase in speed of inoculation in the use of the device. Additionally, the device of this invention may be constructed so as to lessen the chance of accidental contamination of the media. Further, the entire array of nutrients includable within a singular tubular configuration provide ease of reading and lessen the possibility of error arising from misplacing or losing one or more test containers from a given test series. Spattering of organisms as caused by flaming the conventional inoculation needle is eliminated. Also, because the tube itself can be manufactured containing a plurality of media, the need for stocking multiple media is lessened. The tubes can be manufactured and stored in accordance with a classification defining the use to which the tube is to be put, e.g., an enteric tube, Clostridium tube, etc., thus saving storage space.

As still another advantage of the present invention,

any number of containers can be provided limited only only by the number necessary or desirable to conduct a given test. Tubes composed of a sufiicient number of compartments containing the various nutrient combinations necessary to identify diiierent groups of organisms can readily be prepared for use. Of course, where only one test medium is desired, a single container can readily be provided enclosing such medium and capable of penetration with an inoculation instrument. Other advantages and embodiments will be apparent to those in the art.

We claim:

1. In an inoculation device for inoculating test media with microorganism, container wall means defining an enclosure for containing and completely enclosing a test medium, inoculation means having two connected ends whereby movement of one end correspondingly moves the other, said inoculation means extending through said wall means with both said ends outside said enclosure, said inoculation means being withdrawable through said enclosure, and inlet means for introducing the test medium into said enclosure.

2. The device of claim 1 including removable cap means for enclosing one of said ends of said inoculation means outside said enclosure.

3. The device of claim 2 including removable cap means for enclosing the other of said inoculation means outside said enclosure.

4. An inoculation device for inoculating test medium with microorganism which comprises means defining an enclosure, solid test medium Within said enclosure and inoculation means extending through said enclosure in contact with said test medium and terminating in a free end outside said enclosure.

5. In an inoculation device for inoculating test media with microorganism, wall means defining a plurality of enclosures for containing and completely enclosing test media, elongated inoculation means projecting through said wall means and each of the plurality of enclosures and capable of being Withdrawn therefrom, said inoculation means having two connected ends extending outside said enclosures through said Wall means, and inlet means for introducing test medium into each of said enclosures.

6. An inoculation device for inoculating test media with microorganism which comprises a plurality of separate containers, each of said containers defining a separate enclosure for separately containing a test medium, elongated inoculation means slidably extending through said plurality of containers and through each enclosure of said containers, means at a first end of said inoculation means for withdrawing said inoculation means from said enclosures, and means at a second end of said inoculation means for carrying an inoculum, said second end being joined through said inoculation means to said first end, said first and second ends extending outside said enclosures, whereby upon withdrawing said inoculation means by said first end said inoculum is carried by said second end through said enclosures, said containers including inlet means for introducing test media into each of said enclosures and including means for closing said inlet means.

7. The device of claim 6 including removable cap means enclosing the second end of said inoculation means outside said enclosures.

8. The device of claim 7 including removable cap means enclosing the first end of said inoculationmeans outside said enclosures.

9. An inoculation device for inoculating test media with microorganisms which comprises a plurality of cups each having an enclosing side wall and a bottom wall, said cups being aligned along an open line in enclosing relationship with cup bottoms forming covers of next adjacent cups, first cover means over the resulting uncovered cup, solid state test material in each of said cups, an elongated inoculation needle positioned through openings in said first cover means and in the bottom walls of said 6 cups, said inoculation needle extending through the test materials in said cups and projecting from said first cover means and from the bottom of the cup most distant from said first cover means, second cover means enclosing said first cover means and the projection therefrom of said needle, and third cover means enclosing the bottom of said most distant cup and the projection of the needle therefrom.

10. An inoculation device for inoculating test material which in its vertical position corresponds to the structure of an elongated tube having a plurality of horizontally disposed partition members therein in vertical array, sep arate compartments within said tube in vertical array defined by said partition'members for supportingthe test material within the compartments, an opening in each partition capable of slidably receiving an inoculation needle, an inoculation needle extending through each of said openings for contacting the test material and having a handle end extending from one end partition and the other end of said needle extending from the other end partition of the tube, and a cap on each end of said tube enclosing the projecting ends of said inoculation needle.

11. In an inoculation device for inoculating a test medium with microorganism, wall means defining an enclosure for completely enclosing said test medium, said Wall means being penetrable by inoculation means, single inoculation means having a first end and second end for penetrating said wall means in a plurality of penetrations extending through the enclosure defined by said wall means in a position with said ends protruding through the wall means and from the enclosure at spaced locations, and inlet means for introducing the test medium into said enclosure and into contact with said inoculation means while in said position.

12. The device of claim 11 wherein said wall means has openings defining said spaced locations and shaped to secure said inoculation means.

13. A plurality of containers each including a separate chamber capable of containing an inoculation test medium, said plurality of containers being aligned with each other in the form of separate enclosures for completely enclosing test media, and an elongated inoculation means passing through said containers and said chambers in a position impaling each container and chamber, said containers including normally closed openable inlet means for introducing test medium into each chamber for contact with said inoculation means in said position.

14. The inoculation device of claim 13 including test media in solidified state in said enclosures and wherein the test medium in each enclosure is of composition differing from the test medium in each of the other enclosures.

15. The inoculation device of claim 13 including test media in solidified state in said enclosures and wherein said test media are solidified agar based nutrient materials.

16. The inoculation device of claim 13 including test media in solidified state in said enclosures and wherein said test media are metabolic materials.

17. The device of claim 13 wherein the containers include top openings as said inlet means and are vertically aligned, the bottom of each container closing the top opening of an adjacent container.

18. The method of inoculating a test medium which comprises impaling the wall of an enclosure at two spaced positions with an elongated inoculation means of a length greater than the distance between said two spaced positions whereby the inoculation means protrudes at two opposing ends exteriorly of the enclosure, placing and completely enclosing test medium in the enclosure, contacting a first protruding end of said elongated inoculation means with microorganism sample, and withdrawing the inoculation means by the second end thereof while contacting said first end with test medium in said enclosures whereby the inoculation means carries microorganism sample through said container and test medium.

19. The method of claim 18 wherein said enclosure includes a plurality of separate aligned enclosures for sep arately containing test medium.

' 20. The method of'claim 19 wherein said test medium' is in a solidified state.

21. An inoculation device for inoculating test medium with microorganism, which comprises means defining an enclosure, test medium within said enclosure, and inoculation means extending through openings in said first mentioned means and said enclosure in contact with said test medium and terminating in a free end outside said enclosure, said openings in said first mentioned means and said inoculation means being of cooperating shape for substantially containing said test material against leakage from said enclosure.

References Cited by the Examiner UNITED STATES PATENTS OTHER REFERENCES Frobisher, Fundamentals of Microbiology, 5th ed., pp.

171-173 and 203, W. B. Saunders Co.

A. LOUIS MONACELL, Primary Examiner.

15 ABRAHAM H. WINKELSTEIN, Examiner. 

1. IN AN INOCULATION DEVICE FOR INOCULATING TEST MEDIA WITH MICROORGANISM, CONTAINER WALL MEANS DEFINING AN ENCLOSURE FOR CONTAINING AND COMPLETELY ENCLOSING A TEST MEDIUM, INOCULATION MEANS HAVIN TWO CONNECTED ENDS WHEREBY MOVEMENT OF ONE END CORRESPONDINGLY MOVES THE OTHER, SAID INOCULATION MEANS EXTENDING THROUGH SAID WALL MEANS WITH BOTH SAID ENDS OUTSIDE SAID ENCLOSURE, SAID INOCULATION MEANS BEING WITHDRAWABLE THROUGH SAID ENCLOSURE, AND INLET MEANS FOR INTRODUCING THE TEST MEDIUM INTO SAID ENCLOSURE. 